P. Radler*, N. Baranova*, P. Caldas, M. Lopez Pelegrin, M. Loose (2022). In vitro reconstitution of Escherichia coli divisome activation. Nature Communications 13(1). DOI: 10.1038/s41467-022-30301-y
* shared authorship
We systematically studied the behaviour of bacterial-actin FtsA protein from E.coli on supported lipid membrane and in the presence of treadmilling FtsZ filaments. Our results revealed that, in its active state, FtsA can self-interact and dynamically co-treadmill with FtsZ filaments. The formation of FtsA filaments can be triggered by the recruitment of a cytoplasmic peptide from the transmembrane protein FtsN. Our characterisation of the protein dynamics by real-time microscopy was confirmed by a parallel electron microscopy study, which resolved the structure of FtsA filaments in the presence of cytoplasmic FtsN peptide at the lipid interface (Nierhaus T. et al, Nature Microbiology 2022).
V. Hernandez-Rocamora*, N. Baranova*, E. Breukink, M. Loose and W. Vollmer (2021) Real-time monitoring of peptidoglycan synthesis by class A PBPs on the lipid membrane. eLife DOI: eLife.61525
*shared authorship
We have developed an approach to monitor de novo peptidoglycan synthesis on a biomimetic lipid membrane. Our initial work focused on PBP1b, a bifunctional transmembrane peptidoglycan synthase that polymerises the glycan chain and connects adjacent stem peptides by transpeptidation. We reconstituted PBP1b in a polymer-supported biomimetic lipid membrane and were able to monitor its dynamics and activity in real time. We also developed a FRET-based assay to follow in the real-time activity of class A PBPs from Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activators. Considering that peptidoglycan is an essential component of the bacterial cell envelope and a direct target of β-lactams and glycopeptides, our assay not only unravels the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment but can be further developed to be used for high-throughput screening assays.
N. Baranova, P. Radler, V. Hernandez-Rocamora, C. Alfonso, M. Lopez Pelegrin, G. Rivas, W. Vollmer and M. Loose (2020) FtsZ assembles the divisome by a diffusion-and-capture mechanism. Nature Microbiology, DOI: s41564-019-0657-5
To understand how FtsZ treadmilling dynamics can be transmitted across the cellular membrane, we reconstituted a part of the bacterial division machinery from purified components and found that a membrane anchor of FtsZ cytoskeleton FtsA, and two transmembrane proteins, FtsN and FtsQ self-organised with the FtsZ cytoskeleton into chiral rotating rings. Quantitative image analysis confirmed that all membrane proteins could co-migrate with the treadmilling FtsZ cytoskeleton, but despite their collective directional motion, individual proteins followed FtsZ treadmilling via reaction diffusion. This work allowed us to conclude that treadmilling FtsZ filaments creates a moving zone of signalling activity at the division site, transmitting the spatiotemporal information when and where to activate the cell wall synthesis.
RP Richter, NS Baranova, AJ Day, JCF Kwok (2018) Glycosaminoglycans in extracellular matrix organisation: are concepts from soft matter physics key to understanding the formation of perineuronal nets? Current Opinion in Structural Biology DOI: 10.1016/j.sbi.2017.12.002
Conventional wisdom has it that proteins fold and assemble into definite structures, and that this defines their function. Glycosaminoglycans are different. In most cases, the structures they form have a low degree of order, even when interacting with proteins. Here, we discuss how physical features common to all glycosaminoglycans — hydrophilicity, charge, linearity and semi-flexibility — underpin the overall properties of glycan-rich matrices. We integrated soft matter physics concepts (e.g. polymer brushes and phase separation) with our molecular understanding of glycosaminoglycan–protein interactions to comprehend better how glycan-rich matrices assemble, what their properties are, and how they function.
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